Amplification of nucleic acids has revolutionized molecular biology and is now one of the most rapid and versatile methods of producing large quantities of DNA for molecular analysis. Despite the power of this technique, uniform amplification of all templates, regardless of length or G+C content, remains a challenge. Amplification of longer nucleic acid fragments (up to 35 bp) has been accomplished by utilizing different enzymes selected for their thermal stability and editing functions (see PCR Methods and Applications 2:51, 257, 1993; Lundberg et al., Gene 108:1, 1991; Mattila et al., Nucleic Acids Res. 19:4967, 1991; Barnes, Proc. Natl. Acad. Sci. USA 91:2216, 1994; Cheng et al, Proc. Natl. Acad. Sci. USA 91:5695, 1994; Jeffreys et al., Nucleic Acids Res. 16:10953, 1988; Krishnan et al., Nucleic Acids Res. 19:6177, 1991; Maga and Richardson, Biotechniques 11:185, 1991; Rychlik et al., Nucleic Acids Res. 18:6409, 1990; and Kainz et al., Anal. Biochem. 202:46, 1992). However, templates with high G+C content, regardless of length, are only variably amplifiable or sometimes completely unamplifiable even in the presence of reagents that facilitate strand separation, stabilize the polymerase, or isostabilize DNA (e.g., dimethyl sulfoxide (DMSO), formamide, glycerol, or tetramethylammonium chloride (TMACl)) (Bookstein et al., Nucleic Acids Res. 18:1666, 1990; Sarkar et al., Nucleic Acids Res. 18:7465, 1990; Pomp and Medrano, Biotechniques 10:58, 1991; and Hung et al., Nucleic Acids Res. 18:4953, 1990).
Although the average G+C content of the human genome is about 40%, individual genes and genetic elements may have G+C content that is higher. For example, the human c-myc gene is 60% G+C with regions of greater of 70% G+C. As well, the majority of 5' ends of genes and promoters have regions that are G+C rich, and some diseases, such as Fragile X Syndrome, result from the in vivo expansion of G+C rich triplets (e.g., CGG for Fragile X Syndrome). Detection of these triplets in both normal and diseased individuals is difficult. Furthermore, the inability to uniformly amplify DNAs with high G+C content hinders other methods, such as quantitation of transcripts, gene mapping and sequence analysis.
In the present invention, compositions and methods are provided for amplifying nucleic acids regardless of their G+C content as well as providing other related advantages.